Fast-sedimenting DNA Research Articles

Function of Gene 49 of Bacteriophage T4 III, Isolation of Holliday Structures from Very Fast-Sedimenting DNA

Branched DNA molecules were identified in ClaI digests of cytosine containing very fast-sedimenting DNA (VFSDNA CYT) which was isolated from Escherichia coli infected with the multiple mutant 49 -GT7 of phage T4. In about 10% of randomly picked ClaI fragments branches with three arms (Y-structures) as well as four arms (Holliday structures) were seen in the electron microscope. Branched structures were absent from ClaI digests after treatment with purified endonuclease VII (gp49) in vitro.

Growth-Medium-Dependent Repair of DNA Single-Strand and Double-Strand Breaks in X-Irradiated Escherichia coli

The X-ray resistance of logarithmic phase cells of Escherichia coli K-12 is enhanced threefold by growth in rich medium versus minimal medium (N. J. Sargentini, W. P. Diver, and K. C. Smith, Radiat. Res. 93, 364-380, 1983). In this work, X-ray-induced DNA strand breaks were assayed by sedimentation in alkaline and neutral sucrose gradients to correlate the enhanced survival of rich-medium-grown cells with an enhanced capacity for DNA repair. While rich-medium-grown cells showed no enhanced capacity for repairing DNA single-strand breaks in buffer, i.e., fast, polA-dependent repair, they did show an enhanced capacity to repair both single-strand and double-strand breaks in growth medium, i.e., slow, recA-dependent repair. This enhanced capacity for DNA repair in rich-medium-grown cells was inhibited by rifampicin post-treatment, indicating the requirement for de novo RNA synthesis. Kinetic studies indicated that the repair of DNA double-strand breaks was a complex process. Relative to the sedimentation rate in neutral sucrose gradients of nonirradiated DNA, the sedimentation rate of X-irradiated DNA first changed from slow to very fast. Based on alkaline sucrose gradient sedimentation studies, all the strand breaks had been repaired during the formation of the very fast sedimenting DNA. With continued incubation, the sedimentation rate of the DNA on neutral sucrose gradients decreased to the normal rate.

Genetic analysis of very fast sedimenting DNA of bacteriohage T4.

We have previously shown that abnormally fast sedimenting DNA which accumulates upon infection of mutants of T4 phage defective in gene 49 is recombination intermediates, while the mutant gives rise to the recombination deficient phenotype, judging from genetic mating tests under the semipermissible condition (Minagawa and Ryo (1979) Mol, gen. Genet. 170: 113; Minagawa et al. (1983a) Virology 126:138; Miyazaki et al. (1983) Genetics 104:1). To see the step to which the recombination process has proceeded in the DNA, we investigated by the transfection assay whether the generation of contiguous recombinant strands is completed. Such strands were found to be formed normally or nearly normally in the absence of the gene 49 function, supporting the view that the gene product is involved in resolution of branched recombinational intermediates into linear duplexes.

Structural features of very fast sedimenting DNA formed by gene 49 defective T4

Very fast sedimenting DNA (VFS DNA) of T4 phage, which is formed by infection with a mutant in gene 49, was examined by electron microscopy after mild treatment with DNase I. It showed Y-shaped, branched strands in addition to linear strands. Each branch contained a single-stranded interruption about 60 nucleotides long at its proximal end. The average number of branches per T4 DNA unit was close to the average number of sites susceptible to gene 49 nuclease. Both numbers were consistently changed by the addition of a secondary mutation in a gene involving recombination.

Involvement of the bacterial groM gene product in bacteriophage T7 reproduction. I. Arrest at the level of DNA packaging.

The multiplication of bacteriophage T7 is blocked in Escherichia coli M. The genetic determinant of this ability (groM) to inhibit T7 growth was transferred to an E. coli K-12 recipient by means of conjugation. We determined at which precise step T7 maturation is blocked. Phage-directed protein and DNA synthesis as well as degradation of host DNA were not qualitatively affected. Instead of infective phages, only preheads were produced. These, however, were maturable in vitro. The newly synthesized phage DNA accumulated in a concatemeric form and matured from its tetrameric or longer forms (very fast sedimenting DNA) only into its dimeric form (fast-sedimenting DNA) or longer forms. The following step, i.e., the maturation of the dimeric to unit-length DNA, was not observed. Since the concatemeric form of T7 DNA accumulated in spite of the presence of maturable preheads, it is likely that the maturation process was blocked at the level of DNA packaging. As intermediates in the packaging process, we found some prehead-DNA complexes. We interpreted these as true assembly intermediates (or breakdown products thereof), since the attached DNA was still in its concatemeric form. This shows that the very first DNA packaging step, the binding of the progeny DNA to the preheads, was obviously not blocked. Rather, a later step, such as the filling of the preheads with T7 DNA or the stabilization of completely packaged particles (i.e., the final cutting of the concatemers into unit-size length), was inhibited.

Structural Alterations of the DNA in Cerebellar Neurons after Whole-Brain Irradiation

Male Sprague-Dawley rats weighing 260 to 280 g were whole-brain-irradiated with x-ray doses of 433, 867, 1083, 1300, 1516, and 1713 rad. Over the next 2.25 years rats were killed at various times, and the state of the DNA in their cerebellar neurons was examined by sedimentation through alkaline sucrose gradients in reorienting zonal rotors. The data were analyzed as the percentage of the sedimenting DNA with sedimentation coefficients greater than 300 S, an arbitrarily selected category of no defined molecular significance. The general pattern at all doses consisted first of a slow return to the unirradiated DNA state that was relatively dose dependent. This was followed by an increase in the amount of DNA sedimenting >300 S; both the extent and time course of this increase appeared to be dose dependent. Finally, the DNA degraded at a relatively dose independent rate. There was little change in the neuronal DNA from unirradiated rats during this study. The data suggest that increases in the amount of fast-sedimenting DNA observed 30 to 80 weeks after low to moderate doses of whole-brain irradiation represent a type of DNA damage rather than repair and that this damage ultimately results in degradation of the neuronalmore » DNA and death of the rat.« less

Psoralen-DNA Cross-linking Photoadducts in Dyskeratosis Congenita: Delay in Excision and Promotion of Sister Chromatid Exchange

Dyskeratosis congenita is a rare X-linked recessive disease, characterized by mucosal leukokeratosis, nail dystrophy, telangiectasia, reticulated hyperpigmentation, pancytopenia, and a heightened susceptibility to infection and malignancy. We exposed cultured fibroblasts and peripheral leukocytes from normal persons and from 2 unrelated young adult men with dyskeratosis congenita to 4,5',8-trimethylpsoralen and ultraviolet light. We than compared certain of their responses. Labeled DNA from fibroblasts exposed to 4,5',8-trimethylpsoralen and ultraviolet light showed fast-sedimenting DNA, a pattern we interpreted as evidence that cross-linking, psoralen-DNA photoadducts had been formed by the treatment. Fast-sedimenting DNA persisted for 24 hr in dyskeratosis congenita cells but disappeared from normal cells during a 24-hr repair period. Cultured peripheral blood leukocytes from persons with this syndrome similarly exposed to 4,5',8-trimethylpsoralen and ultraviolet light developed more sister chromatid exchanges than did cells from normal persons. These data suggest that a heightened susceptibility in DNA cross-links may be of fundamental importance in the etiology of dyskeratosis congenita.

Genetic control of formation of very fast sedimenting DNA of bacteriophage T4

Formation of very fast sedimenting DNA (VFS-DNA) in cells of Escherichia coli infected with phage T4 carrying a defect in gene 49 was differentially affected by a secondary mutation in gene 30 or 46; a mutation of gene 46 markedly reduced formation of VFS-DNA, whereas that of gene 30 did not.

Substrate specificity of gene 49-controlled deoxyribonuclease of bacteriophage T4: Special reference to DNA packaging

It has been reported that T4 gene 49 controls a nuclease-like activity which attacks very fast sedimenting DNA (VFS-DNA) formed in cells infected with a mutant defective in gene 49 (Frankel, F. R., Batcheler, M. L., and Clark, C. K. (1971) J. Mol. Biol. 62: 439–463). The protein responsible for this activity was partially purified and shown to be an endonuclease. The DNase only attacks T4 DNA having a specific structure; besides VFS-DNA, it cleaves T4 DNA which contains gaps with an internal 5′-phosphorylated terminus. Native, denatured, and nicked T4 DNAs are not susceptible to the enzyme action.

The interaction of dianhydrogalactitol with DNA in cultured yoshida sarcoma cells

Using alkaline sucrose gradient sedimentation centrifugation it was found that treatment of Yoshida sarcoma cells in culture for 1 h with increasing concentrations of dianhydrogalactitol (DAG) enhanced the sedimentation rate of DNA in a dose-dependent manner. There was no difference between the amount of protein which co-sedimented with DNA released from treated and untreated cells. When DNA was extracted from the cells using a p-amino-salicylate-phenol mixture, the protein content of DNA seemed not to be affected by DAG. The possibility that DAG could form interstrand crosslinking in cellular DNA was suggested from renaturation studies. The appearance of a fast sedimenting DNA in the alkaline sucrose gradient and the evidence for a cross-linked DNA detected by renaturation technique, only appeared later than 6 h after treatment. A similar delayed effect on the depression in the rate of DNA synthesis was also observed. These data suggest that the inhibition of DNA synthesis may be related to the delayed formation of DNA interstrand cross-links.

Restriction enzyme cleavage of DNA resulting from gently lysed Escherichia coli

When E. coli or lambda infected E. coli are gently lysed the DNA is released as a very fast sedimenting species that is presumably bound to membrane material. If this complex is now subjected to restriction enzyme cleavage, only a minor fraction of the fast sedimenting DNA remains and this is found, after purification, to be enriched for branched molecules.

Viral DNA synthesis in vitro with nuclei isolated from adeno-associated virus type 1-infected cells

Electron microscope autoradiograms revealed that grains (viral DNA) were found in the nuclei of cells coinfected with adeno-associated virus type 1 (AAV1) and a temperature-sensitive mutant of human adenovirus type 31 (H31 tsA13) at the nonpermissive temperature (40°). Nuclei isolated from cells coinfected with AAV1 and H31 tsA13 at 40° were active in AAV1-DNA synthesis in vitro. Alkaline sucrose gradient analysis of the in vitro products showed that DNA synthesized with nuclei isolated from coinfected cells sedimented faster than marker AAV1-DNA, although DNA synthesized with nuclei isolated from cells coinfected in the presence of cycloheximide cosedimented with the marker DNA. This fast-sedimenting DNA shifted to the position of the marker DNA after treatment with papain, trypsin, and sodium dodecyl sulfate followed by extraction with phenol. This observation suggests the involvement of a protein in the formation of fast-sedimenting viral DNA.

Function of gene 49 of bacteriophage T4. II. Analysis of intracellular development and the structure of very fast-sedimenting DNA

With the exception of mutants in gene 49, all mutants in phage T4 defective in the process of head filling accumulate a normal replicative DNA intermediate of 200S. Mutants in gene 49 produce a very fast-sedimenting (VFS) DNA with s values of greater than 1,000S. The intracellular development of the VFS-DNA generated in gene 49-defective phage-infected cells was followed by sedimentation analysis of crude lysates on neutral sucrose gradients. It was observed that the production of a 200S replicative intermediate is one step in the development of VFS-DNA. After restoring permissive conditions the development of the VFS-DNA can be reversed, but the 200S form is not regenerated under these conditions. The process of head filling can take place from the VFS-DNA under permissive conditions. From the absence of other components in the VFS-DNA complexes, its high resistance to shearing, its resistance against the attack of the single-strand-specific nuclease S1, and from its appearance in the electron microscope, a complex structure of tightly packed DNA is inferred. The demonstration by the electron microscope of branched DNA structures sometimes closely related to partially filled heads is taken in support of the idea that the process of head filling in gene 49-defective phage-infected cells is blocked by some steric hindrance in the DNA. In light of these results, the role of gene 49 is discussed as a control function for the clearance of these structures. A fixation procedure for cross-linking of gene 49-defective heads to the VFS-DNA allowed us to study progressive stages in the process of head filling. Electron microscopic evidence is presented which suggests that during the initial events the DNA accumulates in the vertexes of the head.

Function of gene 49 of bacteriophage T4. I. Isolation and biochemical characterization of very fast-sedimenting DNA.

Very fast-sedimenting DNA was isolated from cells after infection with gene 49 defective phage T4. This DNA appeared membrane bound throughout the time after infection and could be isolated either in the membrane-bound form (M-DNA) or free of membrane (released DNA) depending on the lysis procedure. Released DNA formed complexes of marked stability with sedimentation velocities between 1,400S and 2,100S. These complexes did not seem to contain material other than DNA. This was concluded from the results of RNA, protein, and membrane labeling experiments and density analysis. In addition, these complexes were resistant against treatment with n-butanol, phenol. chloroform-methanol, sodium dodecyl sulfate, Sarkosyl, Pronase, RNase, or lysozyme. The observation that more then 90% of the purified very fast-sedimenting DNA is retrapped by magnesium-Sarkosyl crystals (M-band) suggests that the M-band technique may not be sufficient as a test for DNA-membrane attachment.

Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus

A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus. The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein. The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl. The isolated protein can bind to both circular and linear duplex DNA. Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients. The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template. The binding protein itself is free of detectable DNA polymerase or nuclease activity.

Repair of double-strand breaks in Micrococcus radiodurans.

Micrococcus radiodurans has been shown to sustain double-strand breaks in its DNA after exposure to X-radiation. Following sublethal doses of X-rays (200 krad in oxygen or less), the cells were able to repair these breaks, and an intermediate fast-sedimenting DNA component seemed to be involved in the repair process.

Intracellular forms of adenovirus DNA. 3. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells.

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.

Strandedness and complementarity of DNA from long-term RNA-dependent DNA polymerase reactions of Soehner-Dmochowski murine sarcoma virus.

The DNA product of the endogenously instructed RNA-dependent DNA polymerase reaction of murine sarcoma virus continued to be synthesized for as long as 64 h in the presence of 0.008% Triton X-100. Higher detergent concentrations and actinomycin D inhibited DNA product synthesis. The DNA product from long-term polymerase reactions consisted of small DNA fragments as shown by sedimentation in alkaline sucrose gradients. The enzymatic DNA product was separated into a slow sedimenting fraction and a fast sedimenting fraction by rate-zonal centrifugation. Fast sedimenting DNA was the predominant fraction made in viral polymerase reactions containing 262 mM NaCl. By using a combination of S-1 nuclease and pancreatic RNase A, the amount of single-stranded DNA, double-stranded DNA, and DNA-RNA hybrid present in the slow-sedimenting and fast-sedimenting fractions was determined. Under standard polymerase conditions of 70 mM NaCl, single-stranded DNA was the major form of DNA found in both fractions. In contrast, the prevalent form of DNA made in the presence of 262 mM NaCl was DNA-RNA hybrid. Hybridization studies in which either S-1 nuclease or pancreatic RNase A was used to measure hybrid formation demonstrated not only that the DNA product was complementary in base sequence to the RNA genome, but also that at least 79 to 84% of the RNA genome was transcribed into complementary DNA.

The rate of DNA replication in the polytene chromosomes of Rhynchosciara angelae

The distribution pattern of 5-bromodeoxyuridine-labelled DNA from salivary glands of Rhynchosciara angelae upon caesium chloride gradient centrifugation was studied with DNA of different molecular weights. This pattern suggests a very low rate of DNA chain growth in polytene chromosomes. The rate of DNA chain growth was found to be 0.025 μm/min at 24 °C. The result was obtained through the development of a mathematical expression which took into account the distribution of the 5-bromodeoxyuridine-labelled DNA in CsCl gradients. DNA pulse-labelled for a short time sediments more slowly in alkaline sucrose gradients than DNA which has been labelled during a prolonged incubation. However, in neutral sucrose gradients the pattern of banding is the same for both DNAs. This indicates a discontinuity in the newly synthesized DNA strand, but not in the template strand. The transition of slow sedimenting to fast sedimenting DNA observed in alkaline sucrose gradients, occurs very slowly, as would be expected for a slow rate of DNA chain growth. The data obtained provided a means of comparing the number of replication points with the rate of DNA chain elongation and the length of S phase in Rhynchosciara.

A sedimentation analysis of DNA found in Escherichia coli infected with phage λ mutants

A study was made of the phage DNA produced in Escherichia coli infected with mutants of λ unable to produce infectious particles because of defects in genes controlling head formation. Bacteria were irradiated with UV to eliminate host DNA synthesis and then infected with various λ sus mutants under conditions where subsequent radioactive labelling appeared exclusively in phage DNA. The intracellular DNA was extracted and examined by sedimentation in neutral and alkaline sucrose gradients. Mutants in genes A, B, C, D, and E cause blocks in DNA maturation which result in the accumulation of “rapidly sedimenting” species. Mutants in genes W and F accumulate much less of the rapidly sedimenting DNA and synthesize about 50–100% of the normal amounts of mature molecules as found in control infections. Intracellular phage DNA was purified from host material by density labelling and examined by electron microscopy. The “rapidly sedimenting” species appeared to be linear concatamers with lengths up to and exceeding four units. In an A mutant circular molecules were also found; these were both of monomer and multimer length (dimers and trimers). The proportion of immature DNA found by electron microscopy (long molecules) and by sedimentation (fast sedimenting DNA) agreed very well.